DirectN-terminal sequence analysis of rat liver plasma membrane glycoproteins separated by two-dimensional polyacrylamide gel electrophoresis

Abstract

Nine previously uncharacterized membrane glycoproteins from normal rat liver have been analyzed by amino acid sequencing from two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) after transblotting to Immobilon‐P membranes. Three of these components show altered levels of expression in liver tumors. A single electroblotted polyacrylamide gel yielded sufficient quantities of these glycoproteins for amino acid sequencing and the N‐terminal structure could be determined for four of them. The remaining five glycoproteins of interest were not sequenceable in this manner, presumably because they had blocked N‐termini. Prior to electrophresis, two enrichment methods were applied to the crude liver membrane preparations: affinity chromatography with concanavalin A to isolate the plasma membrane glycoproteins and then fast protein liquid chromatography on Superose 12 to obtain components having a specific range of molecular weights. These materials were next subjected to 2‐D PAGE using pH 4–6 carrier ampholytes in the first dimension and 7.5% sodium dodecyl sulfate gels in the second. The proteins were then electroblotted to Immobilon‐P membranes and located by staining with Coomassie Brilliant Blue R‐250. Our results demonstrate that N‐terminal sequencing (gas‐phase) can be achieved on polypeptides obtained from approximately 250 μg of total glycoproteins applied to a single 2‐D gel.